The long-term objective of this proposal is to elucidate the roles, mechanisms and functions of sphingolipids in cell regulation and pathogenesis, focusing on the opportunistic fungus Cryptococcus neoformans. This fungal pathogen mainly infects immunocompromised patients, and it is the most common cause of fungal meningitis worldwide. The ability of C. neoformans to grow at high temperatures is required to produce infection. During infection, this fungus also possesses the ability to produce virulence factors, such as melanin, which mainly protects fungal cells from the immune response. Inositol phosphoryl ceramide synthase 1 is a fungus-specific enzyme encoded by the essential IPC1 gene. It regulates the formation of complex sphingolipids, the levels of phytoceramide and sphingoid bases, and diacylglycerol (DAG). Therefore, the regulation of Ipc 1 might represent an important factor in the maintenance of crucial cellular functions. In the case of a fungal opportunistic pathogen, the impairment of Ipc 1 activity could exert a significant impact on host-fungus interaction, either by altering the cell adaptation to host-temperature or/and by modifying the production of virulence factor(s). In our previous studies, we regulated the expression of the essential IPC1 gene by using the GAL7 promoter and showed that C. neoformans is no longer pathogenic in an animal model of cryptococcal meningitis when Ipcl is down-regulated. In our ongoing epistasis analysis to understand how Ipcl regulates pathogenicity, we find that: 1) high temperature environments differentially regulate Ipcl through the modulation of phytoceramide or/and sphingoid bases; and 2) Ipc 1 regulates melanin production through the modulation of DAG and the consequent activation of protein kinase C (Pkcl). Based on these observations, we hypothesize that Ipc 1 is a key regulator of pathogenicity of C. neoformans by modulating virulence factors, such as the response to high temperatures and melanin production, by controlling the level of sphingolipids, phytoceramide and DAG. Thus, we will: 1) determine the role and mechanism of Ipc 1 in heat stress; and 2) establish the role of Ipcl in melanin formation. These studies not only will establish novel functions for the lipids regulated by Ipcl in the pathogenicity of C. neoformans, but will also allow defining new targets, such as Ipc 1 and Pkcl, for the development of new drugs for the treatment of human fungal infections.